This study explores if radiation and erythropoietin (Epo) synergize to alter the self-renewal of breast cancer stem cells (BCSCs) and to reprogram differentiated breast cancer (BC) cells into induced breast cancer stem cells (iBCSCs). Using established cell lines and a unique imaging system for breast cancer stem cells, patient-derived tumor samples and clinical pretreatment blood samples of BC patients undergoing radiation treatment (RT) it will explore if Epo acts on irradiated BCSCs directly, if it alters the BCSC nich, and which pathways are engaged during radiation-induced reprogramming of differentiated BC cells in the presence of Epo. In a prospective clinical study we will test, if high serum Epo level correlate with high BCSC burden, nodal status and local control after breast-conserving surgery (BCS) and RT. We hypothesize, that ionizing radiation reprograms differentiated breast cancer cells into radioresistant iBCSCs and that endogenous Epo synergizes with radiation to increase the BCSCs pool, thereby impairing radiation therapy outcome. Limited stage BC is usually treated with mastectomy or BCS and RT. For RT, low hemoglobin (Hb) levels predict early treatment failure. Attempts to increase Hb levels using Epo led to inferior outcome of BC patients, suggesting that signaling through the Epo receptor promotes tumor growth. Recent studies indicate that BCs are organized hierarchically with a small population of BCSCs, capable of re-growing a tumor while their progeny lack this feature. According to the cancer stem cell (CSC) hypothesis, cure is only possible if all CSCs are eliminated, suggesting that the deleterious effects of Epo were mediated by effects of Epo on BCSCs. Because of the rare nature of BCSCs, the effects of Epo on these cells have not been investigated. BCSCs are relatively radioresistant. Furthermore, EpoR expression on tumor cells is inversely correlated with prognosis and rhEpo and RT increases the number of BCSCs in vitro possibly through RT-induced reprogramming of differentiated BC cells into iBCSCs. The hypothesis will be tested by 1) investigating the effects of Epo and RT on BCSCs, 2) exploring the pathways activated during reprogramming, and 3) testing if endogenous Epo affects BCSCs clinically. Cancer-related anemia is a condition with a high incidence in cancer patients. This proposal will study if erythropoietin activates pathways that synergize with radiation to increase the pool of therapy-resistant BCSCs and evaluate if endogenous Epo level correlate with BCSC burden and clinical outcome. The long-term goals of this proposal are to study under which circumstances Epo interferes with BCSC numbers and treatment outcome. Understanding the pathways may lead to novel combined treatments that will improve BC treatment outcome.